(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
28 November 2002 (28.11.2002)
(10) International Publication Number
PCT WO 02/095355 A2
(51) International Patent Classification 7 : G01N
(21) International Application Number: PCT/US02/15998
(22) International Filing Date: 17 May 2002 (17.05.2002)
(25) Filing Language: English
(26) Publication Language: English'
(30) Priority Data:
09/860,661
18 May 2001 (18.05.2001) US
(63) Related by continuation (CON) or continuation-in-part
(CIP) to earlier application:
US 09/860,661 (CIP)
Filed on Not furnished
(71) Applicant (for all designated States except US):
THERASENSE, INC. [US/US1; 1360 South Loop
Road, Alameda, CA 94502 (US).
(72) Inventor; and
(75) Inventor/Applicant (for US only): HELLER, Adam
[US/US]; 5317 Valburn Circle, Austin, TX 78731 (US).
(74) Agents: WU, Louis, L. et ah; Reed & Associates, Suite
2 1 0, 800 Menlo Avenue, Mcnlo Park, C A 94025 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
VN, YU, ZA, ZM, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR,
GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
NE, SN, TD, TG).
Published:
— without international search report and to be republished
upon receipt of that report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
5 (54) Title: ELECTROCHEMICAL METHOD FOR HIGH-THROUGHPUT SCREENING OF MINUTE QUANTITIES OF CAN-
1- DID ATE COMPOUNDS
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o
-10
(57) Abstract: An electrochemical method is provided for successively assessing the efficacy of each of a plurality of candidate
compounds by determining the degree to which each compound directly or indirectly affects the rate of a target molecule-catalyzed
electrochemical reaction. The method involves successively introducing samples containing candidate compounds into the detection
chamber of a flow cell sized to contain up to about lOOul liquid, measuring the rate of the target molecule-catalyzed electrochemical
reaction, flushing the flow cell with a carrier after each measurement, and determining the efficacy of each candidate compound
based on the measured rate of the target molecule-catalyzed electrochemical reaction. The method is useful for detecting very small
quantities of potential active agents, on the order of 0.1 pg. ideally, a nonleachable redox mediator is disposed on the working
electrode so as to facilitate transfer of electrons between the target molecule and the working electrode surface. The method may be
employed to assess the capability of candidate compounds as enzyme inhibitors or as ligands, e.g., as receptor-binding ligands.
WO 02/095355 PCT/US02/15998
Electrochemical Method for High-Throughput
Screening of Minute Quantities of Candidate Compounds
Technical Field
This invention relates to methods for screening compounds to assess their
potential utility as biologically and/or chemically active agents. More particularly, the
invention relates to an electrochemical process and flow cell device for conducting such
screening methods with very small samples, containing at most about one nanogram of a
candidate compound. Candidate compounds include potential pharmacologically active
agents such as enzyme inhibitors and receptor-binding ligands, as well as biologically or
chemically active agents useful in agricultural products.
Background Art
Combinatorial processes have made possible the synthesis of very large
numbers of candidate compounds that need to be screened for their activity, e.g., as
enzyme inhibitors, as receptor-binding ligands, or the like. In general, these processes
may involve parallel synthesis of diverse compounds by sequential addition of reagents
that leads to the generation of large chemical libraries having molecular diversity. Thus,
combinatorial chemistry typically involves the systematic and repetitive, covalent
connection of a set of different "building blocks" of varying structures to yield large arrays
of diverse candidate compounds. Such combinatorial processes have been disclosed in a
number of patents. See, e.g., U.S. Patent Nos. 5,982,387 to Hollinshead and 5,859,190 to
Meyer et al.
Since combinatorial synthesis produces very large numbers of candidate
compounds in quantities that are quite small, there is a need in the art for a rapid, high-
throughput screening method capable of evaluating very large numbers of compounds in
minuscule sample sizes.
Previous screening methods have involved assessing enzyme inhibition using
dissolved enzymes, or photonic, usually colorimetric, analysis of a probed solution when
the enzyme is immobilized. The activity is determined through measuring the time
dependence of the concentration of a substrate of the enzyme, meaning its depletion from
the solution. Alternatively, the accumulation of the product of the enzyme-catalyzed
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reaction may be determined. The specific binding of a candidate ligand to a receptor is
usually measured by labeling the candidate blocking agent with a fluorescent tag and
observing the luminescence of the immobilized, often membrane-bound, receptor. These
methods do not, however, lend themselves to a rapid, high-throughput screening technique
useful with minuscule quantities of candidate compounds.
High-throughput screening assay systems in microscale fluidic devices have
been proposed. U.S. Patent No. 6, 1 50, 1 80 to Parce et al, for example, describes
microfluidic devices that may be employed to identify inhibitors of an enzymatic reaction
through fluorescence. Sample flow in such microfluidic devices may be induced by
applying voltages to various reservoirs in the devices to effect electroosmosis of
electrokinesis. However, this means that the usefulness of this system is limited to the
analysis of certain sample fluids in devices having correspondingly functionalized
channels. In addition, such functionalized channels may be fouled with repeated use,
further limiting analytical capacity of this type assay.
Thus, there is a need to for an improved method for evaluation of the
biological activity of candidate compounds.
Disclosure of the Invention
Accordingly, it is a primary object of the invention to address the
aforementioned need in the art by providing a method for rapidly and efficiently assessing
the biological and/or chemical activity of each of a plurality of candidate compounds in
succession, wherein the quantity of each candidate compound in any given sample is
extremely small, on the order of 0. 1 pg to about 1 ng.
It is another object of the invention to provide such a method using a flow cell
containing a detection chamber having a capacity in the range of about 0.5 to about 100
jxL, and wherein the aggregate volume of the liquid flowing through the system at any
given time is accordingly in the range of about 0.5 to about 100 jjL.
It is a further object of the invention to provide such a method wherein the
time required for sample introduction, electrochemical measurement, and flushing of the
detection chamber is on the order of 30 seconds or less.
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It is another object of the invention to provide such a method wherein the
activity assessed is the ability of each candidate compound to affect the catalytic activity
of a target molecule.
It is still another object of the invention to provide such a method wherein the
target molecule is an enzyme and the candidate compounds are potential inhibitors of the
enzyme.
It is a further object of the invention to provide such a method wherein the
activity assessed is the ability to bind to a ligand-binding partner such as a receptor, and
the candidate compounds are potential ligands.
It is a further object of the invention to provide such methods in which such
assessment is accompanied with the accuracy and precision associated with
electrochemical analytical techniques.
Additional objects, advantages and novel features of the invention will be set
forth in part in the description which follows, and in part will become apparent to those
skilled in the art upon examination of the following, or may be learned by practice of the
invention.
In a first embodiment, then, an electrochemical method is provided for
successively assessing the efficacy of each of a plurality of candidate compounds. The
method involves use of an electrochemical flow cell having a detection chamber adapted
to contain in the range of about 0.5 jal to about 100 \il of liquid, an inlet for directing a
stream of liquid into the detection chamber, and an outlet for directing liquid out of the
detection chamber. The detection chamber includes a working electrode and a reference
electrode, and, disposed on the working electrode, a target molecule that catalyzes an
electrochemical reaction of at least one reactant. For each candidate compound that is
evaluated, the efficacy assessed is the capability of the compound to directly or indirectly
affect the catalytic activity of the target molecule, which can be determined by the degree
to which the presence of a particular candidate compound affects the rate at which the
electrochemical reaction proceeds. Initially, about 0.5 \il to about 100 pi of a sample is
introduced through an inlet into the detection chamber so as to contact the target molecule.
The sample is a liquid medium containing about 0.1 pg to about 1 ng of a candidate
compound Following sample introduction, the rate of the electrochemical reaction is
measured, either amperometrically (e.g., by measuring current generated at the working
electrode) or coulometrically. After the measurement, the detection chamber is flushed
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with a carrier liquid, which is allowed to flow through the detection chamber for a time at
least sufficient to ensure that the chamber is substantially free of the candidate compound.
The aforementioned steps » sample introduction, electrochemical measurement, and
carrier liquid flush — are repeated with each of the plurality of candidate compounds in
succession, with the time required for each cycle not exceeding about 30 seconds. The
efficacy of each candidate compound is determined from the electrochemical
measurement made with respect to that compound (or from the average of a plurality of
electrochemical measurements made* with respect to that compound), either during each
cycle or following a plurality of cycles.
In a related embodiment, the candidate compound is a candidate ligand evaluated
for its ability to bind to a ligand-binding partner, e.g., a receptor. Thus, in this case, the
aforementioned method is modified so as to provide an electrochemical method for
successively assessing the ability of each of a plurality of candidate ligands to bind to a
ligand-binding partner. The method involves use of an electrochemical flow cell as
described above, i.e., a flow cell having a detection chamber adapted to contain in the
range of about 0.5 pi to about 100 \i\ of liquid, an inlet for directing a stream of liquid into
the detection chamber, and an outlet for directing liquid out of the detection chamber. The
detection chamber includes a working electrode and a reference electrode, and, disposed
on the working electrode, a ligand-binding partner having an initial ligand bound thereto
and, attached to the initial ligand, a redox enzyme that catalyzes an electrochemical
reaction of a substrate of the redox enzyme. For each candidate ligand that is evaluated,
the efficacy assessed is the capability of the ligand to displace the initial ligand from the
ligand-binding partner, which can be determined by the decrease in the observed rate of
the electrochemical reaction. That is, a candidate ligand that does not have affinity for the
ligand-binding partner will not affect the observed rate of the electrochemical reaction,
while a candidate ligand that has some affinity for the ligand-binding partner will reduce
the observed rate of the electrochemical reaction to some degree (proportional to the
candidate ligand's affinity for the ligand-binding partner), and a candidate ligand that has a
strong affinity for the ligand-binding partner will substantially reduce the observed rate of
the electrochemical reaction or even eliminate the occurrence thereof. To carry out this
method, a sample having a volume in the range of about 0.5 |al to about 100 pi containing
about 0,1 pg to about 1 ng of a candidate ligand is introduced through an inlet into the
detection chamber. Following sample introduction, the rate of the electrochemical
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reaction is measured, either amperometrically or coulometrically, as above. After the
measurement, the detection chamber is flushed with a carrier liquid, followed by
introduction of another sample into the detection chamber. The steps of sample
introduction, electrochemical measurement, and carrier liquid flush are repeated with each
of the plurality of candidate ligands in succession, with the time required for each cycle
not exceeding about 30 seconds.
In another embodiment, an alternative electrochemical method is provided for
successively assessing the ability of each of a plurality of candidate ligands to bind to a
ligand-binding partner such as a receptor. This method involves a "direct" assay instead of
a "competitive" assay as just described. Accordingly, this alternative method involves (a)
providing a flow cell as before, comprised of a detection chamber adapted to contain 0.5
\il to about 100 pi of liquid, an inlet for directing a stream of liquid into the detection
chamber, and an outlet for directing liquid out of the detection chamber, the detection
chamber including a reference electrode and a working electrode with the ligand-binding
partner disposed thereon; (b) introducing about 0.5 jil to about 100 \i\ of a sample
containing about 0. 1 pg to about 1 ng of a candidate ligand through the inlet into the
detection chamber, wherein the candidate ligand is bound to a redox enzyme that catalyzes
an electrochemical reaction of a substrate of the redox enzyme; (c) determining whether or
not the electrochemical reaction is taking place; (d) flushing the detection chamber as
described above; and (e) assessing the efficacy of the candidate ligand from the
determination made in step (c). That is, if the candidate ligand binds to the binding
partner, the electrochemical reaction will be apparent on the surface of the working
electrode. A carrier liquid is introduced as before, and the process may be repeated with
each of a plurality of candidate ligands in succession. Again, the cycle time is on the order
of 30 seconds or less.
Brief Description of the Drawings
The invention is described in detail below with reference to the following
drawings, wherein like reference numerals indicate corresponding elements throughout the
several views.
FIG. 1 illustrates in exploded view an electrochemical flow cell for use in
conjunction with the method of the present invention.
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FIG. 2 illustrates in cross sectional view the detection chamber formed within
the electrochemical flow cell of FIG. 1.
FIG. 3 illustrates in exploded view an alternative electrochemical flow cell for
use in conjunction with the present invention.
5
Modes for Carrying Out the Invention
I. Terminology and Definitions:
Before describing the present invention in detail, it is to be understood that this
10 invention is not limited to particular candidate compounds, target molecules, redox
mediators, or flow cell designs, as such may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
It must be noted that, as used in this specification and the appended claims, the
15 singular forms "a," "an" and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to "a target molecule" includes a
combination of two or more target molecules, reference to "a redox mediator" includes
combinations of two or more redox mediators, reference to "a reactant" includes two or
• more different reactants, and the like.
20 In describing and claiming the present invention, the following terminology
will be used in accordance with the definitions set out below.
"Amperometry" includes steady-state amperometry, chronoamperometry, and
Cottrell-type measurements.
The term "attach" as used herein refers to either covalent or noncovalent
25 binding. Noncovalent binding will typically involve "adsorption" such as may occur
through hydrogen bonding, van der Waal's forces, polar attraction or electrostatic forces
(i.e., through ionic bonding), or the like.
The term "candidate" as in "candidate compound" and "candidate ligand,"
refers to a compound or ligand, respectively, that may or may not have the desired efficacy
30 or activity that is assessed using the present methods. The present method is capable of
assessing the efficacy of very small quantities of a candidate compound, on the order of
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0.1 pg to about 1 ng, preferably about 0.1 pg to about 100 pg, more preferably about 0.1
pg to about 1 0 pg, and ideally about 0.1 pg to 1 pg.
"Coulometry" is the determination of charge passed or projected to pass during
complete or nearly complete electrolysis of a compound, either directly on the electrode or
5 through one or more electron transfer agents. The charge is determined by measurement of
charge passed during partial or nearly complete electrolysis of the compound or, more
often, by multiple measurements during the electrolysis of a decaying current and elapsed
time. The decaying current results from the decline in the concentration of the electrolyzed
species caused by the electrolysis.
10 A "counter electrode" refers to one or more electrodes paired with the working
.electrode, through which passes an electrochemical current equal in magnitude and
opposite in sign to the current passed through the working electrode. The term "counter
electrode" is meant to include counter electrodes that also function as reference electrodes
(i. e. a counter/reference electrode) unless the description provides that a "counter
15 electrode" excludes a reference or counter/reference electrode.
The "detection chamber" is defined herein as a region of the flow cell sized to
contain only that portion of the sample that is to be interrogated during an assay.
An "effective diffusion coefficient" is the diffusion coefficient characterizing
transport of a substance, for example, a candidate compound, a candidate ligand, an
20 analyte, an enzyme, or a redox mediator, in the volume between the electrodes of the
electrochemical cell. In at least some instances, the cell volume may be occupied by more
than one medium (e.g., the sample fluid and a polymer film). Diffusion of a substance
through each medium may occur at a different rate. The effective diffusion coefficient
corresponds to a diffusion rate through this multiple-media volume and is typically
25 different than the diffusion coefficient for the substance in a cell filled solely with sample
fluid.
"Electrolysis" is the electrooxidation or electroreduction of a compound either
directly at an electrode or via one or more electron transfer agents (e. g., redox mediators
and/or enzymes).
30 The term "facing electrodes" refers to a configuration of the working and
counter electrodes in which the working surface of the working electrode is disposed in
approximate opposition to a surface of the counter electrode. In at least some instances,
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the distance between the working and counter electrodes is less than the width of the
working surface of the working electrode.
The "flow cell" of the invention is a device configured to allow flowthrough of
very small quantities of a liquid sample, on the order of 0.5 \d to 100 preferably about
5 0.5 pi to about 10 \il 9 most preferably about 0.5 pi to about 1 pi, in order to detect the
presence of and/or measure the concentration of a candidate compound in liquid sample
via electrochemical oxidation and reduction reactions. These reactions are
transduced to an electrical signal that can be correlated to an amount or concentration of
the candidate molecule.
10 A compound is "immobilized" on a surface when it is entrapped on or
chemically bound to the surface.
An "indicator electrode" includes one or more electrodes that detect partial or
complete filling of a detection chamber and/or measurement zone.
The term "ligand" refers to a molecular segment or an intact molecule,
15 generally an intact molecule, capable of attaching to a ligand-binding partner, e.g., a
receptor, through either covalent or noncovalent binding.
Conversely, a ligand "binding partner" refers to a molecular segment or intact
molecule, generally an intact molecule that is capable of binding a ligand either covalently
or noncovalently.
20 A "nondiffusible," "nonleachable," or "non-releasable" compound is a
compound that does not substantially diffuse away from the working surface of the
working electrode for the duration of the electrochemical screening assay.
"Potentiometry" and "chronopotentiometry" refer to taking a potentiometric
measurement at one or more points in time.
25 A "redox mediator" is an electron transfer agent for carrying electrons between
a compound and the working electrode, either directly or indirectly.
A "reference electrode" includes a reference electrode that also functions as a
counter electrode (i. e., a counter/reference electrode) unless the description provides that
a "reference electrode" excludes a counter/reference electrode.
30 "Sorbent material" is material that wicks, retains, and/or is wetted by a fluid
sample and that typically does not substantially prevent diffusion to the electrode.
WO 02/095355 PCT/US02/15998
A "surface in the detection chamber" includes a surface of a working
electrode, counter electrode, counter/reference electrode, reference electrode, indicator
electrode, a spacer, or any other surface bounding the detection chamber.
• A "working electrode" is an electrode at which candidate compound is
electrooxidized or electroreduced with or without the agency of a redox mediator.
A "working surface" is that portion of a working electrode that is covered with
a nonleachable redox mediator and exposed to the sample, or, if the redox mediator is
diffusible, a "working surface" is that portion of the working electrode that is exposed
These reactions are transduced to an electrical signal that can be correlated to an amount
or concentration of the candidate molecule.
The "cycle time" is the time required to introduce the sample, to perform the
measurement, and to restore the system to readiness for accepting a new sample.
n. Overview:
The invention provides an electrochemical technique for conducting high-
throughput screening of a plurality of candidate compounds in rapid succession, with a
cycle time on the order of 30 seconds or less. The candidate compounds may be, for
example, enzyme inhibitors, ligands (including receptor-binding ligands), or other
compounds capable of directly or indirectly reacting with a target molecule wherein
reaction with the target molecule results in an electrochemically detectable event, e.g., an*
increase or decrease in current. As described below, many of these candidate compounds-
are pharmacologically active agents.
The method involves use of an electrochemical flow cell capable of detecting
extremely small quantities of a candidate compound in a sample. The volume of the
sample introduced into the flow cell will not exceed about 100 and may be less than
about 1 0 jil or even less than about 1 pi. The amount of candidate compound contained in
each sample introduced into the detection chamber is at most about 1 ng, but may be less
than about 100 pg, and may be less than about 10 pg, or even less than about 1 pg.
Generally, although not necessarily, the minimum volume of sample is about 0.5 pi and
the minimum quantity of candidate compound in each sample, i.e., the minimum quantity
that can be accurately and consistently detected using the present method, is about 0.1 pg.
Depending upon the molecular weight of the candidate compound, quantities that are at
most about 10 pmoles, preferably at most about 1 pmole, and most preferably about 0.1
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pmole, can be detected. The capability of the present invention to detect such small
quantities of a candidate compound is in part a result of the sensing chemistry used and in
part a result of the flow cell structure and dimensions. That is, the flow cell generally
comprises a thin film cell containing a detection chamber structured and sized to contain a
5 volume of a liquid medium that is less than about than about 1 00 pi and that may be less
than about 10 pi or even less than about 1 pi (as indicated above), which volume gives rise
to a thin film that is less than about 100 pm in height, optimally less than about 50 pm in
height.
The method further involves providing a flowing sample stream through a
10 detection chamber of a flow cell. The flow is stopped periodically, and the concentration
of the candidate compound is determined by an electrochemical method, such as
coulometry. After the measurement, flow is resumed, thereby removing the sample the
participated in the measurement from the flow cell. Alternatively, sample may flow
through the chamber at a very slow rate, such that all of the candidate compound is
15 electrolyzed in transit, yielding a current dependent only upon candidate compound
concentration and flow rate. The construction of flow cells that may be used to carry out
the inventive method and their operation are detailed below.
Enzyme inhibitors represent one class of candidate compounds that can be
screened using the present method. Inhibitors of any enzyme can be screened, including
20 both redox enzymes, i.e., oxidoreductases, and non-redox enzymes. The enzymes may .
thus be in any of the six internationally recognized enzyme classes, i.e.: oxidoreductases,
which catalyze the transfer of electrons, hydride ions or hydrogen atoms; transferases,
which catalyze transfer of molecular groups or segments; hydrolases, which catalyze
hydrolysis reactions (transfer of functional groups to water); lyases, which catalyze the
25 reaction or formation of double bonds; isomerases, which catalyze the transfer of atoms or
groups within molecules so as to convert one isomeric form to another; and ligases, which
catalyze the formation of new covalent bonds, e.g., carbon-carbon bonds. By way of
example, and not limitation, inhibitors of the following specific enzymes can be assessed
using the present techniques (the enzymes are listed along with their corresponding
30 substrates and reaction product):
WO 02/095355
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PCT/US02/15998
HjISjZj X JYLCf
li AlMt L. AK I
SUBSTRATE(S)
CVT7A4T>T A D V DD ATM TPT/C\ ll
glucose oxidase
glucose
electrons
choline oxidase
choline
electrons
acetylcholine esterase
acetylcholine
choline
uncase
urate
electrons
cholesterol oxidase
cholesterol,
electrons
oxalate oxidase
oxalate,
electrons
superoxide dismutase
UUn radicals
H 2 0 2
horseradish peroxidase
tt r~\
H 2 U 2
electron vacancies, electrons
consumed
xanthine oxidase
xanthine
electrons
alkaline phosphatase
p-aminophenyl phosphate
p-aminophenol
phosphodiesterase
bis-p-aminophenyl phosphate
p-aminophenol
lactate dehydrogenase
lactate pyruvate, NAD or
XT A TAT T i
NADH
NADH, NAD, electrons or
electron vacancies
formate dehydrogenase
formate, NAD
NADH
glucose-6-phosphate
dehvdro en a<?e
glucose-6-phosphate, NADP
NADPH
6-phosphogluconate
dehydrogenase
gluconate-6-phosphate, NADP
NADPH
pyruvate kinase
phosphonoenol pyruvate, ADP
pyruvate, ATP
The candidate compounds may also be ligands, screened in terms of their
capability to bind to ligand-binding partners including but not limited to receptors.
Receptors, as is well known in the art, are cellular macromolecules to which a compound
5 binds in order to initiate its effects. The more studied drug receptors include, for example,
cellular proteins whose normal function is to act as receptors for endogenous regulatory
ligands, e.g., hormones, growth factors, neurotransmitters, and autacoids. Examples of
specific receptors include, by way of illustration: plasma-bound protein kinases, which are
receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor
10 (PDGF), and certain lymphokines; cell-surface guanylyl cyclase, which is a receptor for
atrial natriuretic peptides (ANP), guanylin and pheromones; neurotransmitter receptors, or
ion channels, which are receptors for substances such as garnma-aminobutyric acid,
glutamate, aspartate and glycine; G protein-coupled receptors, which facilitate binding of
GTP to specific GTP-binding proteins (or "G proteins"); and transcription factors, which
15 are receptors for steroid hormones, thyroid hormone, vitamin D, and retinoids.
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Candidate ligands may also be screened for activity to bind to ligand-binding
partners other than receptors. For example, a ligand may be screened for its activity as an
antigen, to bind to an antibody, or conversely, as an antibody, to bind to an antigen.
Binding of a candidate ligand to a particular binding partner (or binding of a candidate
5 binding partner to a particular ligand) is an integral part of immunoassays, protein-binding
assays, nucleic acid hybridization assays, and amplification assays. In typical
immunoassays, as noted above, the ligand may be either an antigen or an antibody and the
corresponding binding partner is an antibody or antigen, respectively. Various alternative
immunoassay, hybridization and amplification techniques and formats thereof are well
10 known in the art.
The sample containing the candidate compound or ligand may be derived from
a biological source and will be in the form of a liquid medium in which the candidate
compound is dissolved, dispersed or suspended. The liquid medium may, for example, be
water, blood or urine.
15
DDL The Flow Cell:
A flow cell is required to carry out the electrochemical assessment method of
the invention. Depending on the desired electrochemical assessment, flow ceil
construction may differ. However, all flow cells of the invention are comprised of a
20 detection chamber having a volume capacity in the range of about 0,5 pi to 100 jil,
preferably about 0.5 pi to about 10 |xl, most preferably about 0.5 pi to about 1 pi. An inlet
is provided for directing a stream of liquid into the detection chamber, and an outlet is
provided for directing liquid out of the detection chamber. The detection chamber also
includes a working electrode, a counter electrode, and an optional reference electrode.
25 When only two electrodes are used, the counter electrode also serves as a reference
electrode. The electrodes serve to carry out the electrochemical assessment method of the
invention.
It should be noted that the sensing chemistry and the construction of the flow .
cell both influence the accuracy and precision of electrochemical assessment, as discussed
30 below in detail. However, as a general rule, rapid transport of species is desirable for the
flow cell. Thus, cells recognized for their fast mass transport, e.g., falling film cells, may
be advantageously employed. In addition, the flow through the cell can be continuous or
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periodically stopped. When periodically stopped, the cell is referred to a "stopped flow
cell."
An example of an electrochemical flow cell suitable for use in carrying out the
aforementioned method is illustrated in FIG. 1 . As is the case with all figures referred to
5 herein, FIG. 1 is not to scale, and, in particular, certain dimensions may be exaggerated for
clarity of presentation. In the figure, the flow cell is shown generally at 10 as comprised
of a working block 12 and an auxiliary block 14 each constructed of non-conducting
materials such as plastics (e.g., polymethyl methacrylate, polycarbonate, polyethylene
terephthalate, polystyrene, polyimide, poly(dimethylsiloxane), polypropylene or other
10 polyolefins, etc.). As illustrated, the blocks are generally comprised of solid cylinders
with planar base surfaces. For example, auxiliary block 14 is illustrated as having a
generally planar base surface 16, a radially outwardly facing cylindrical side surface 18,
and a generally planar axially inwardly facing mating surface 20 facing generally ring-
shaped gasket 22. Working block 12 is similarly constructed.
15 Auxiliary block 14 includes a counter electrode 24 and a reference electrode
26, respectively coupled to a counter electrode lead 28 and a reference electrode lead 29.
In an alternative embodiment, a single electrode may be present on the inner, mating
surface of the auxiliary block, coupled to a single electrode lead; in such a case, the
counter electrode doubles as the reference electrode. Auxiliary block 14 also houses inlet
20 tube 30 terminating in an inlet opening 32 on mating surface 20 and, correspondingly, an
outlet opening 34 in fluid communication with outlet tube 36. Working block 12 includes
a working electrode 38 that is generally circular in cross-section and enters the working
block at approximately the center 40 of outer surface 42 of the working block and
continues through the block, terminating in a generally disk shape on the inner mating
25 surface 44 of the working block. The exposed area of the working electrode on the mating
surface 44 of the working block, which becomes one surface, generally although not
necessarily the bottom surface, of the flow cell's detection chamber upon assembly of all
components, is typically less than about 10" 3 cm 2 , preferably less than about 10" 4 cm 2 ,
more preferably less than about 10" 5 cm 2 .
30 - The components of the flow cell are assembled by coupling the working block
to the auxiliary block with the gasket 22 therebetween and holding the assembly together
in fluid-tight alignment. One means for reversibly maintaining the assembly in fluid-tight
alignment is depicted in FIG. 1 , wherein axially extending apertures 46 and 48 in the
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working block are respectively aligned with apertures 50 and 52 in the gasket and with
apertures 54 and 56 in the auxiliary block. Apertures 54 and 56 are preferably threaded so
that bolts 58 and 60 can be inserted through the working block, the gasket and the
auxiliary block to hold the components of the flow cell together.
5 As illustrated in FIG. 2, assembly of the flow cell components in this manner
results in the formation of a detection chamber 62 having a height equivalent to the
thickness of the gasket 22, and bounded by the interior surface of working block 12
(including the exposed terminus of working electrode 38), the interior surface of auxiliary
block 14, and the radially inwardly facing surface 64 of the gasket. As may be seen, a
10 flow path is provided through the detection chamber in the form of a linear path, such that
sample fluid introduced into the detection chamber through inlet tube 30 flows across the
surface of the working electrode 38 to the outlet aperture that serves as an entrance to
outlet tube 36.
It is to be understood that the aforementioned flow cell configuration and
15 associated method of use is for the purpose of illustration, and that a variety of flow cell
configurations may be employed. In addition, the flow of the liquid medium introduced
into the detection chamber is not necessarily linear, but may be radial or have other types
of flow paths.
FIG. 3 illustrates an alternative flow cell that can be used in conjunction with
20 the present method. In this figure, the flow cell is shown generally at 66 as comprised of a
working block 68 and an auxiliary block 70 each constructed of non-conducting materials
as described above with respect to the working and auxiliary blocks of FIG. 1. Working
block 68 has a generally planar base surface 72, a radially outwardly facing cylindrical
side surface 74, and a generally planar axially inwardly facing mating surface 76 facing
25 generally ring-shaped gasket 78. As before, auxiliary block 70 is similarly constructed.
Working block 68 includes a working electrode 80 and a reference electrode
, 82, respectively coupled to a working electrode lead 84 and a reference electrode lead 86.
In an alternative embodiment, a single electrode may be present on the inner, mating
surface of the working block, coupled to a single electrode lead. Auxiliary block 70
30 houses counter electrode 88, coupled to counter electrode lead 90, and is provided with a
central substantially cylindrical passageway 92 that serves as an inlet tube; the flow of
liquid is shown at the arrow 94. The auxiliary block also houses outlet tube 96 terminating
in an outlet opening 98 on mating surface 100 of the auxiliary block.
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As with the flow cell illustrated in FIG. 1, the various components are
assembled by coupling the working block to the auxiliary block with the gasket 102
therebetween and holding the assembly together in fluid-tight alignment. One means for
reversibly maintaining the assembly in fluid-tight alignment is as depicted in FIG.l,
5 wherein axially extending apertures 104 and 106 in the working block are respectively
aligned with apertures 108 and 110 in the gasket and with apertures 112 and 114 in the
auxiliary block. Each aperture is preferably threaded so that bolts can be threaded through
the auxiliary block, the gasket, and the working block as in FIG. 1 .
It will be appreciated that since the fluid inlet in this embodiment terminates in
10 the center of gasket 102, the fluid flow in the assembled flow cell will be in a direction
that is generally radially outward from the center of the detection chamber formed
between the auxiliary and working blocks.
IV. Flow Cell Operation:
15 A flow cell, either as described above or constructed according to an
alternative flow cell design known in the art, is used for successively assessing: (1) the
efficacy of a plurality of candidate compounds to affect the catalytic activity of a target
molecule, or (2) the ability of each of a plurality of candidate ligands to bind to a ligand-
binding partner. In either case, the rate or simply the occurrence of an electrochemical
20 reaction that takes place within the detection chamber of the flow cell is used to assess the
activity of a candidate compound or ligand. However, there are notable variations with
respect to flow cell operation, depending on the type of candidate compound (enzyme
inhibitor, ligand, etc.), the screening technique used (e.g., a direct assay versus a .
competitive assay), and the molecular moieties used to carry out the process (enzymes,
25 ligand-binding partners, competitive ligands, etc.).
In assessing the capability of candidate compounds to affect the catalytic
activity of a target molecule, a sample comprised of a liquid medium having a volume of
less than about 100 pi, or less than about 10 pi, or even less than 1 jil, containing at most 1
ng, typically less than about 100 pg, or even less than about 10 pg or even less than about
30 1 pg of a candidate compound, is introduced into the detection chamber of a suitable flow
cell through an inlet. A target molecule that catalyzes an electrochemical reaction of one
or more reactants is disposed on the working electrode. The target molecule is preferably
nonleachable but may be diffusible from the electrode in certain cases. Either during flow,
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or when flow is stopped, the rate of the electrochemical reaction occurring at the working
electrode is measured, so that the effect of the candidate compound on the rate of reaction
may be determined relative to a baseline Corresponding to the rate of the reaction in the
absence of a candidate compound. The effect of a candidate compound on the rate of the
5 electrochemical reaction in turn correlates to the ability of the candidate compound to
affect the catalytic activity of the target molecule. Thus, for example, a candidate
compound that is an inhibitor of the target molecule's catalytic activity will decrease the
rate of the electrochemical reaction. After measurement of the electrochemical reaction
rate, the detection chamber is flushed by employing a suitable carrier liquid to force the
10 sample-containing liquid medium out of the detection chamber through the outlet and into
the outlet tube. Continuous flow of the carrier liquid through the detection chamber is
provided for a time at least sufficient to ensure that the chamber is substantially free of the
candidate compound. The aforementioned steps may be repeated with each of a number
of candidate compounds in rapid succession.
15 Since the electrochemical reaction catalyzed by the target molecule involves
the chemical transformation of one or more reactants, i.e., compounds that serve as
substrates for the catalytic reaction, the operation of the flow cell in this embodiment
requires such a reactant so that the electrochemical reaction catalyzed by the target
molecule can occur. A sufficient amount of reactant should be maintained in the
20 detection chamber in order to provide a baseline signal generated by the electrochemical
reaction. When the method is employed to assess the efficacy of a candidate compound in
enhancing the rate of the electrochemical reaction, a sufficient supply of reactant should
be present in order to compensate for an enhanced rate of reaction in the presence of
certain candidate compounds. In order to ensure an adequate supply of reactant through a
25 plurality of successive cycles, the reactant is preferably introduced into the detection
chamber when the sample is introduced. This often involves dissolving or suspending the
reactant in the liquid medium of the sample. However, the timing and the manner of
reactant introduction into the detection chamber are not critical. In some instances, the
reactant may be disposed within the chamber before the introduction of the sample.
30 It should be noted that the reactant must be chosen according to the catalytic
capability of the target molecule. Thus, for example, when the target molecule is a redox
enzyme, the reactant will be a substrate of the enzyme. Upon exposure to the candidate
compound, the enzyme's catalytic activity may be enhanced or suppressed. In the latter
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case, when a candidate compound inhibits the enzymatic activity of the target, molecule,
the inhibition of the electrochemical reaction may be observed amperometrically as a drop
in current or coulometrically as a drop in charge. Conversely, reaction acceleration is
observed with catalytic activity enhancement. Thus, when the flow cell is used to screen
5 the efficacy of candidate compounds to affect the catalytic activity of a redox target
molecule, e.g., a redox enzyme, the rate of the electrochemical reaction of the reactant
may be directly affected.
However, when the flow cell is used to screen the efficacy of candidate
compounds to affect the catalytic activity of a non-redox moiety, e.g., a non-redox
10 enzyme; the compounds' catalytic activity is assessed in an indirect manner. In this case,
the reactant selected serves as a substrate for the non-redox enzyme (typically disposed on
the working electrode), which catalyzes a reaction resulting in a reaction product that
serves as a substrate for the redox enzyme, i.e., as a substrate for the target molecule. A
cascade of enzymes may be used in this manner, with the initial enzyme transforming the
15 reactant into a reaction product that serves as a substrate for a second enzyme, which then
transforms the substrate into a reaction product that serves as a substrate for a third
enzyme, and so forth. The rate of the last reaction in the cascade, caused by the target
molecule's catalytic action as a redox enzyme, is dependent on the rate of the first reaction,
and thus reflects the ability of a candidate compound to affect the catalytic activity of the
20 first enzyme. Thus, turnover of the non-redox moiety is measured through a change in the
concentration of its ultimate product, i.e., the final reactant produced by the cascade,
which is electrolyzed (electrooxidized or electroreduced) in a reaction involving the target
molecule.
More specifically, to screen the efficacy of a candidate compound to affect the
25 catalytic activity of a non-redox moiety, a sample comprised of a liquid medium
containing the candidate compound may be introduced into the detection chamber of a
flow cell. The target molecule is disposed on the working electrode as well as the non-
redox moiety; both the non-redox moiety and the target molecule are preferably
nonleachable from the electrode. A sample comprised of a liquid medium containing a
30 candidate compound is introduced into the detection chamber. The target molecule
disposed on the working electrode serves to catalyzes an electrochemical reaction of a
reactant However, the reactant is not independently introduced or provided in the
detection chamber. Instead, the reactant is produced through chemical and/or
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electrochemical reactions through the catalytic assistance of the non-redox moiety.
Therefore, in this embodiment, one or more precursors must be provided in the detection
chamber in order to assess the efficacy of each candidate compound to affect the catalytic
activity of the non-redox moiety. If the candidate compound is highly effective in
5 suppressing the catalytic activity of the non-redox moiety, then the precursor(s) will not be
converted into the reactant and the electrochemical reaction involving the target molecule-
catalyzed reaction of the reaction will not take place. Conversely, if the candidate is
ineffective in suppressing or effective in promoting the catalytic activity of the non-redox
moiety, a reactant will be formed from the precursors. In turn, the target will catalyze the
10 electrochemical reaction of the reactant. As described above, the efficacy of the
compound may be assessed either during flow, or when flow is stopped. A carrier liquid
may then be used to force the sample-containing liquid medium out of the detection
chamber through the outlet and into the outlet tube, and allowing continuous flow of the ,
carrier liquid through the detection chamber for a time at least sufficient to ensure that the
15 chamber is substantially free of the candidate compound. As a result, the aforementioned
steps may be repeated with each of a number of candidate compounds in rapid succession,
whether or not the candidate compounds directly or indirectly affect the catalytic activity
of the target molecule.
In addition, the flow cell may be used for successively assessing the ability of
20 each of a plurality of candidate ligands to bind to a ligand-binding partner. Such
assessment can be carried out by employing either a ligand displacement technique or a
ligand binding technique. In ligand displacement, a flow cell is provided as before,
comprising a detection chamber that includes a working electrode, a counter electrode and
an optional third electrode, and is used in conjunction with a small amount of sample
25 containing a correspondingly small amount of candidate compound, with typical, possible
and preferred amounts as described previously. Here, however, a redox enzyme,
preferably a nonleachable redox enzyme, is disposed on the working electrode, wherein
the redox enzyme catalyzes an electrochemical reaction of a substrate. Attached to the
redox enzyme is an initial ligand having a ligand-binding partner bound thereto (e.g., a
30 receptor having its natural ligand bound thereto). When a sample comprising a liquid
medium containing a candidate ligand is introduced through the inlet into the detection
chamber, the efficacy of the candidate ligand is assessed by determining the degree to
which the rate of the electrochemical reaction is affected at the working electrode. That is,
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if the candidate ligand binds to the receptor more strongly than .the initial ligand, the initial
ligand will be displaced, reducing the rate of (or even entirely eliminating) the
electrochemical reaction taking place at the working electrode surface. A decrease in the
rate of the electrochemical reaction thus indicates the ability of a candidate ligand to bind
5 to the ligand-binding partner, with a significant decrease in reaction rate indicating that a
particular candidate ligand has substantial affinity for the receptor binding ligand. A
carrier liquid is introduced to flush the detection chamber as described previously, i.e., for
a time at least sufficient to ensure that the chamber is substantially free of the candidate
ligand. The foregoing steps may then be repeated with each of a plurality of candidate
10 ligands in succession in a high-throughput screening process, with at most 30 seconds
. between the introduction of each candidate ligand into the cell
In contrast to the aforementioned ligand displacement technique, which is
essentially a competitive assay, a ligand binding technique is a direct assay that may also
be used in conjunction with the present method. In this technique, the detection chamber
15 of the flow cell includes a working electrode with the ligand-binding partner disposed
thereon. Each sample is comprised of a liquid medium containing a candidate ligand,
wherein, in this embodiment, the candidate ligand is bound to a redox enzyme that
catalyzes an electrochemical reaction of a substrate. The sample is introduced through the
inlet into the detection chamber, and the efficacy of the candidate ligand is assessed by
20 determining whether or not the electrochemical reaction is taking place at the working
electrode. That is, if the candidate ligand binds to the binding partner, the electrochemical
reaction resulting from the presence of the redox enzyme on the working electrode will be
apparent on the surface of the working electrode. The flow cell is flushed with a carrier
liquid as before, and the process may be repeated with each of a plurality of candidate
25 ligands in succession in a high-throughput screening process, with at most 30 seconds
between the introduction of each candidate ligand into the cell.
Depending on the type of analysis and the particular analytical reagents that
are used, the electrochemical reaction that is monitored occur at different electrode
potentials or under different flow conditions. In some instances, the electrochemical
30 reaction may occur upon application of approximately the same potential to the working
and reference electrodes. In other cases, the electrochemical reaction may take place upon
the application of a potential across the working and reference electrodes. In addition, as
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discussed above, electrochemical measurements may be taken either when the sample is
flowing through the detection chamber or during a sample flow interruption.
V. The Working Electrode:
5 It should be apparent from the above description of the operation of the flow
cell that the working electrode is a critical component of the flow cell for carrying out the
inventive method. In general, the working electrode is small Thus, the working surface
of the electrode is also small. When circularly shaped, the electrode is typically less than
about 1 mm in diameter and preferably less than 0.1 mm diameter. In addition, the
10 working electrode may be formed from a molded carbon fiber composite. Carbon fiber
composite electrodes are-preferred when short measurement times are sought. Usually the
diameter of the individual fibers is 1-20 pm, typically about 5-10 pm. When these are
spaced such that the center-to-center distance between neighboring fibers is greater than
about 10 times their diameter the capacitance is greatly reduced and the measurement
15 times are short, typically of less than about 1 sec. The electrode may alternatively be
comprised of an inert non-conducting base material, such as polyester, upon which a
suitable conducting layer is deposited. The conducting layer typically has relatively low
electrical resistance and should be substantially electrochemically inert over the potential
range of the flow cell during operation. Suitable conducting layers include gold, carbon,
20 platinum, ruthenium dioxide, palladium, and conductive epoxies, such as, for example,
Eccocoat® CT5079-3 Carbon-Filled Conductive Epoxy Coating (available from W. R.
Grace Company, Woburn, Massachusetts), as well as other non-corroding materials
known to those skilled in the art. To form this type of electrode, the conducting layer is
deposited on the surface of the inert material by any conventional method such as
25 chemical vapor deposition or printing.
The working electrode is constructed to allow for electrical connection to
external electronics such as a voltage source or current measuring equipment by providing
a lead or a tip located outside the detection chamber. However, other methods or
structures (such as contact pads) may also be used to connect the working electrode to the
30 external electronics.
As described above, the working electrode must have disposed thereon a target
molecule, e.g., an enzyme, and/or a ligand-binding partner. In order to monitor the
electrochemical reaction that takes place at the flow cell, the target molecule and/or the
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ligand-binding partner must be electrically connected to the working electrode to allow
electron transfer therebetween. The target molecule and/or the ligand-binding partner are
typically provided in a layer that coats the working electrode. Preferably, the target
molecule is nonleachable from the electrode or immobilized with respect to the polymeric
5 layer. Covalent attachment to the polymeric layer is preferred. Optionally, the reactant
involved in the reaction catalyzed by the target molecule may be included in the polymeric
layer as well. This electrocatalytic coating layer may further comprise an electron
conducting redox mediator that establishes an electrical connection between the electrode
and the target molecule and/or the ligand-binding partner. The electron-conducting redox
10 mediator is preferably comprised of a polymer having one or more redox species ionically,
covalently or coordinatively bound thereto, in which case the redox mediator is termed a
"redox polymer." The polymer may or may not be the polymer of the aforementioned
coating containing the target molecule and/or the ligand-binding partner. The inclusion of
an electron conducting redox polymer is a unique feature because it may swell in the
15 presence of the liquid medium (generally water or an aqueous solution) containing the
candidate compound and thus enhance mass transport of liquid soluble matter such as ions
and reactants. The liquid solubility of such matter in the redox polymer at about 25 °C
should be greater than about 0.1 mM, preferably greater than about 1 mM and optimally
greater than about 10 mM. Such redox polymers are described, infra.
20 When the present method is to screen the efficacy of candidate compounds in -
affecting the catalytic activity of a non-redox moiety, a plurality of compounds must be
used in combination to produce a cascade of reactions, wherein the last reaction in the
series is the electrochemical reaction catalyzed by the redox enzyme that serves as the
target molecule. In this embodiment, the working electrode is constructed to effect and
25 exploit such a cascade of reactions, as described in detail in part (IV). As before, the
working electrode may be coated with a polymeric layer containing the target molecule.
Again, covalent attachment to the polymeric layer is preferred. The non-redox moiety is
preferably nonleachably disposed or immobilized on the working electrode as well, as are
the various intermediate compounds, e.g., enzymes, involved in the cascade.
30 To prevent electrochemical reactions from occurring on portions of the
working electrode not coated by the redox mediator when a nonleachable mediator is used,
a dielectric material may be deposited on the electrode over, under, or surrounding the
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region(s) on which the redox mediator is disposed. Suitable dielectric materials include, by
way of example, waxes and non-conducting organic polymers such as polyethylene. The
dielectric material may also cover a portion of the redox mediator on the electrode.
Generally, the surface regions containing the redox mediator will be covered in a manner
that prevents exposure of the redox mediator to the sample.
VI. The Counter Electrode:
The counter electrode may be constructed in a manner similar to that described
above for the working electrode. The counter electrode may also be a counter/reference
electrode. Alternatively, a separate reference electrode may be provided in contact with
the detection chamber. Suitable materials for the counter/reference or reference electrode
include Ag/AgCl or Ag/AgBr printed on a non-conducting base material or silver chloride
on a silver metal base. For example, a chlorided tip of a silver wire may be used as a
reference electrode. The same materials and methods may be used to make the counter
electrode as are available for constructing the working electrode, although different
materials and methods may also be used. A counter electrode lead allows for convenient
connection to the external electronics, such as a coulometer, potentiostat, or other
measuring device. Since the counter electrode must pass an electrochemical current equal
in magnitude and opposite in sign to the current passed through the working electrode, it
should be designed to allow the counter electrode to accommodate the same or greater
current density than the current density capability of the working electrode.
VDL The Redox Mediator:
In addition to the working electrode, sensing chemistry materials are provided
for the analysis of the candidate compound. As discussed above, the sensing chemistry
materials must be selected according to the type of assessment. Thus, for example, when
the present method is used for successively assessing the ability of each of a plurality of
candidate compound to affect the catalytic activity of a target molecule, the sensing
materials include a target molecule, a reactant, and a carrier liquid. In addition, the
sensing chemistry materials preferably include a redox mediator. The redox mediator may
be diffusible but is preferably nonleachable (i.e., nondiffusible) because successive
introduction of the carrier liquid may reduce the amount of redox mediator in the detection
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chamber thereby compromising the accuracy and the precision of the electrochemical
measurements
The redox mediator, whether it is diffusible or nonleachable, serves to mediate
a current between the working electrode and the target molecule to enable electrochemical
5 analysis as provided herein. That is, the mediator provides electrical contact between the
target molecule and the working electrode. Although any organic or organometallic redox
species can be used as a redox mediator, one type of suitable redox mediator is a transition
metal compound or complex. Examples of suitable transition metal compounds or
complexes include osmium, ruthenium, iron, and cobalt compounds or complexes. In
10 addition, metallocene derivatives, such as, for example, ferrocene, can be used.
Appropriate redox mediators can be identified by one of ordinary skill in the art through
routine experimentation, and/or are described in the pertinent texts and literature
. references. See, for example, International Patent Publication No. WO 00/20626 to
Feldman et al., assigned to Therasense, Inc.
15 The redox mediator may be a redox polymer, i.e., a polymer bound to a redox
species, wherein the redox polymer is provided as a layer on the working electrode.
Preferably, the redox species is nonleachable from the polymer as a result of ionic,
covalent, or coordinative binding thereto. For example, a redox species composed of a
transition metal complex may be nonleachably attached to a polymer in order to form a
20 redox polymer composed of a polymeric transition metal complex. Typically, the
polymers used to form redox polymers contain nitrogen-containing heterocycles, typically
five- or six-membered aromatic heterocycles such as pyrrolyl, pyrrolidinyl, pyridinyl,
quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, such as pyridinyl,
imidazolyl, or derivatives thereof. Thus, suitable polymers for complexation with redox
25 species, such as transition metal complexes as described above, include, for example,
polymers and copolymers of poly (N-vinyl imidazole) (referred to as "PVI") and poly
(4-vinyl pyridine) (referred to as M PVP"), as well as polymers and copolymers of poly
(acrylic acid) or polyacrylamide that have been modified by the addition of pendant
nitrogen-containing heterocycles. Modification of poly(acrylic acid) may be carried out
30 by reaction of some or all of the carboxylic acid functionalities with, for example, an
aminoalkylpyridine or an aminoalkylimidazole, such as 4-ethylaminopyridine, to form
amide linkages. Suitable copolymer substituents of PVI, PVP, and poly(acrylic acid)
include acrylonitrile, acrylamide, acrylhydrazide, and substituted or quaternized 1 -vinyl
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imidazole. The copolymers can be random or block copolymers. As is the case with redox
mediators, appropriate redox polymers are known in the art and can be identified by one of
ordinary skill in the art through routine experimentation.
The redox mediators and/or polymer layers can be bound or otherwise
5 immobilized on the working electrode to prevent leaching of the mediator into the sample.
Immobilization may be accomplished by fiinctionalization of the electrode surface and
then chemical bonding, often covalent chemical bonding, of the redox polymer to the
functional groups on the electrode surface. In addition, the redox mediator can be
otherwise disposed or immobilized on the working electrode by known methods, for
10 example, via formation of multiple ion bridges with a countercharged polyelectrolyte, by
covalent attachment of the redox mediator to a polymer on the working electrode, by
entrapment of the redox mediator in a matrix that has a high affinity for the redox
mediator, or through bioconjugation of the redox mediator to a compound bound to the
working electrode. A variety of methods may be used to immobilize a redox polymer on
15 an electrode surface. One method is adsorptive immobilization. This method is particularly
useful for redox polymers with relatively high molecular weights.
The molecular weight of a redox polymer may be increased, if desired, by
cross-linking. For example, the redox polymer may contain functional groups such as, for
example, hydrazide, amine, alcohol, heterocyclic nitrogen, vinyl, allyl, and/or carboxylic
■20 acid groups, that serve as reactive sites and thus enable crosslinking, using a crosslinking
agent or a second polymer having reactive groups that can bind to the functional groups of
the first polymer. Alternatively or additionally, the functional groups may be added by a
reaction, such as, for example, quaternization of amine-containing polymers to give
■positively charged quaternary ammonium moieties. One example of such a process is the
25 quaternization of P VP with bromoethylamine groups.
Suitable cross-linking agents include, for example, molecules having two or
more epoxide (e. g., poly (ethylene glycol) diglycidyl ether (PEGDGE)), aldehyde,
aziridine, alkyl halide, and azide functional groups or combinations thereof. When a
polymer has multiple acrylate functions, it can be crosslinked with a di-or polythiol; when
30 it has multiple thiol functions it can be crosslinked with a di- or polyacrylate. Other
examples of cross-linking agents include compounds that activate carboxylic acid or other
acid functional groups for condensation with amines or other nitrogen compounds. These
cross-linking agents include carbodiimides or compounds with active
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N-hydroxysuccinimide or imidate functional groups. Yet other examples of cross-linking
agents are quinones (e. g., tetrachlorobenzoquinone and tetracyanoquinodimethane) and
cyanuric chloride. Still other cross-linking agents may also be used. In some
embodiments, an additional cross-linking agent is not required. Further discussion and
5 examples of cross-linking and cross-linking agents are found in the following U.S.
Patents: 5,262,035; 5,262,305; 5,320,725; 5,264,104; 5,264,105; 5,356,786; and
5,593,852.
Thus, it should be apparent that one or more of the various sensing chemistry
materials may be attached to one another and/or to the working electrode by employing
10 the aforementioned techniques. Analogous methods of "attaching" molecular moieties to
each other may be used in sample preparation. For example, one embodiment of the
invention requires a sample containing a candidate ligand bound to a redox enzyme that
catalyzes an electrochemical reaction. In such a case, the candidate ligand can be bound to
the redox enzyme using coupling techniques as described above or other techniques
15 known in the art. For covalent binding, the preferred and most versatile technique
involves the reaction of a nucleophilic moiety on the ligand (e.g., an amino, hydroxyl, or
sulfhydryl group) with an electrophilic moiety on the enzyme (e.g., an aldehyde, an
isocyanate, etc.), or reaction of a nucleophilic moiety on the enzyme with an electrophilic
moiety on the ligand.
20 It should be further noted that in cases wherein certain sensing chemistry
materials are leachable, either a sufficient amount of the leachable material must be
provided when the screening method is initiated or the material must be replenished as
assessment progresses.
25 VIII. Engineering Considerations:
The present method is preferably carried out with a thin layer electrochemical
flow cell structured so as to provide desirable mass transport to the working electrode and
effective utilization of the sample in the detection chamber of the cell. The cell can have
two electrodes, a working electrode and a reference electrode (which also serves as the
30 counter electrode; in this case, it may also be referred to as a "reference/counter
electrode"), or it can have three electrodes, a working electrode, a counter electrode, and a
separate reference electrode. The preferred location of the working electrode is in the thin
layer cell. The reference/counter electrode (in a two-electrode system) or both the
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reference and counter electrodes (in a three-electrode system) can be upstream or
downstream from the working electrode, although the preferred location of these
electrodes is downstream of the working electrode, so that the chemical flux from the
reference and counter electrode reactions will not reach the working electrode. The thin
5 layer cell can be built with working and reference electrodes facing each other Such a
configuration may be achieved by providing an electrically insulating spacer of a
predetermined thickness between the two electrodes. These spacers are typically less than
about 200 Jim and preferably less than about 100 |xm in thickness. The electrodes do not
need, however, to directly face each other. For example, the reference electrode may be
10 positioned adjacent to the outlet such that it is in a downstream position relative to the
flow of sample through the detection chamber. Because the currents are small, less than
10" 5 amperes, the voltage drops are usually not significant and the reference/counter
electrode (or separate reference and counter electrodes) can be located upstream or
downstream. Either the working or the reference electrodes may serve as an indicator
15 electrode used to confirm that the cell is full. In the alternative, an additional electrode
may be provided to detect partial or complete filling of a detection chamber and/or
measurement zone.
It is to be understood that while the invention has been described in
conjunction with the preferred specific embodiments thereof, that the foregoing
20 description as well as the examples which follow are intended to illustrate and not limit the
scope of the invention. Other aspects, advantages and modifications within the scope of
the invention will be apparent to those, skilled in the art to which the invention pertains.
Example 1
25 A system with a total volume of 0. 1 mL comprising a flow cell is constructed.
The cell comprises a detection chamber having an interior volume of 5 (xl, an inlet for
directing a stream of liquid into the detection chamber, and an outlet for directing liquid
out of the detection chamber. The detection chamber contains a working electrode for
assessing the capability of candidate compounds for inhibiting the catalytic activity of
30 horseradish peroxidase, a carbon paste counter electrode, and downstream from the
working electrode, near the outlet, a standard calomel reference electrode. The working
electrode is formed by co-depositing on a conductive substrate horseradish peroxidase and
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a redox polymer formed of poly(4-vinyl pyridine) partially reacted with 2-
bromoethylamine and partially complexed with [Os(bpy) 2 Cl] +/2+ where bpy=2,2'-
bipyridine complex of osmium (2+/3+) and poIy(4-vinyl pyridine). The horseradish
peroxidase and the redox polymer are crosslinked on the substrate by periodate oxidation,
5 whereby aldehyde functions are produced in the oligosaccharides of the peroxidase, and
by adding PEGDGE, polyethylene glycol diglycidyl ether.
An aqueous pH 7 phosphate buffer comprises 0.15 M NaCl and 1 mM
hydrogen peroxide. Three liquid samples are prepared, each sample 0.1 mL. The first
sample contains only the liquid carrier, the second sample contains the liquid carrier and
10 100 pg sodium cyanide, and the third carrier contains the liquid carrier and 100 pg sodium
azide.
The first sample is introduced into the flow cell through the inlet, and the
working electrode is poised at +0.1 V versus the potential of the standard calomel
electrode. The working electrode catalyzes the electroreduction of hydrogen peroxide to
15 water, generating a current by the reaction 2H 4 " + 2e" + H 2 0 2 -> 2H 2 0. The electric current
flowing between the electrodes provides a baseline against which enzymatically inhibited
current is measured.
Then, the second sample is introduced into the flow cell through the inlet,
forcing the first sample out of the outlet of the flow cell. The cyanide combines with
20 heme centers of the horseradish peroxidase and thereby inhibits its catalytic activity. As a
result, the electroreduction of hydrogen peroxide to water is inhibited, and the current
passing from one electrode to another is thereby decreased. The reduction in current
indicates the capability of cyanide to inhibit the catalytic activity of the horseradish
peroxidase.
25 The first sample is introduced again into the flow cell through the inlet,
thereby forcing the second sample out of the outlet of the flow cell. As cyanide is flushed
out of the flow cell, the current between electrodes is increased to its original magnitude.
The return of the current to its original magnitude indicates the complete removal of the
second sample from the detection chamber. The entire cycle time is shorter than 30
30 seconds.
Then, the third sample is introduced into the flow cell. Similar to the case
wherein the second sample is introduced into the detection chamber, the azide in the third
sample combines with heme centers of the horseradish peroxidase and thereby inhibits the
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catalytic activity of the peroxidase. Thus, the third sample also inhibits the
electroreduction of hydrogen peroxide to water, and the current flowing from one
electrode to the other electrode is decreased. The decrease in current indicates the
capability of cyanide to inhibit the catalytic activity of the horseradish peroxidase. In
addition, the difference in the capability of the cyanide and azide to inhibit the catalytic
capability of horseradish peroxidase is evaluated by comparing the difference between the
current passed between the electrodes as the second and third samples are introduced into
the detection chamber.
Example 2
A flow cell as described in Example 1 is provided except that additionally and
nonleachably attached to the working electrode are acetylcholine esterase and choline
oxidase. An aqueous carrier liquid is prepared in an oxygen-containing atmosphere to
comprise the buffer of Example 1 and acetylcholine, , the total volume of the liquid being
less than 0.1 mL. Two samples are prepared. The first sample contains only the buffered
acetylcholine solution; the second sample contains the liquid carrier and 100 pg malathion,
an insecticide.
The first sample is introduced into the flow cell through the inlet, and the
working electrode is poised at +0. 1 V versus the standard calomel electrode. The
acetylcholine in the solution is hydrolyzed by the esterase on the electrode, producing
choline, which, in turn, combines with dissolved oxygen to form hydrogen peroxide. The
hydrogen peroxide is electroreduced to water. An electric current is produced between the
electrodes as a result. The current produced provides a baseline against which current
produced as a result of enzymatic inhibition is measured.
Then, the second sample is introduced into the flow cell through the inlet,
forcing the first sample out of the outlet of the flow cell. The malathion inhibits the
catalytic activity of the acetylcholine esterase. As a result, the production of hydrogen
peroxide is inhibited, and the cmrent passing from one electrode to another is thereby
decreased. The reduction in current indicates the capability of malathion to inhibit the
catalytic activity of the acetylcholine esterase.
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Example 3
A flow cell as described in Example 1 is provided except that the horseradish
peroxidase is replaced with a biological receptor. A first solution contains pH 7 phosphate
buffered 0.15 M NaCl and 1 mM hydrogen peroxide and also a 100 pg of a first peptide
labeled with horseradish peroxidase. When the sample is introduced into the flow cell
through the inlet, the working electrode is held at +0.1 V versus the calomel reference
electrode. If the horseradish peroxide labeled peptide binds with the biological receptor the
hydrogen peroxide is electroreduced and a base-line current flows. .
An second solution is now prepared comprisingthe above buffered hydrogen peroxide
solution, but containing in addition to the horseradish peroxidase labeled first peptide also
1 00 pg of a second un-labeled test-peptide. A decrease in the current indicates affinity of
the second test peptide for the biological receptor.
An third solution is now prepared comprising the above buffered hydrogen
peroxide solution, not containing in addition to the horseradish peroxidase labeled first
peptide but only 100 pg of the second un-labeled test-peptide. A decrease in the current
confirms the affinity of the second test peptide for the biological receptor.
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Claims :
1. An electrochemical method for successively assessing the efficacy of each of a
plurality of candidate compounds, comprising:
5 (a) providing a flow cell comprised of a detection chamber adapted to contain in
the range of about 0.5 \il to about 100 pi of liquid, an inlet for directing a stream of liquid
into the detection chamber, and an outlet for directing liquid out of the detection chamber,
the detection chamber comprising a working electrode and a reference electrode, and,
disposed on the working electrode, a target molecule that catalyzes an electrochemical
10 reaction of a reactant, wherein the efficacy assessed is the capability of each candidate
compound to directly or indirectly affect the catalytic activity of the target molecule,
thereby directly or indirectly affecting the rate at which the electrochemical reaction
proceeds;
(b) introducing about 0,5 pi to about 1 00 jxl of a sample through the inlet into the
15 detection chamber so as to contact the target molecule, wherein the sample is comprised of
a liquid medium containing about 0.1 pg to about 1 ng of a candidate compound; ■
(c) measuring the rate of the electrochemical reaction;
(d) introducing a carrier liquid through the inlet into the detection chamber so as
to force the sample-containing liquid medium out of the detection chamber through the
20 outlet, and allowing flow of the carrier liquid through the detection chamber for a time at
least sufficient to ensure that the chamber is substantially free of the candidate compound;
(e) repeating steps (b), (c) and (d) with each of the plurality of candidate
compounds in succession, wherein each cycle comprised of steps (b), (c) and (d) has a
cycle time not exceeding about 30 seconds; and
25 (f) determining the efficacy of each candidate compound from the measurements
made in (c).
2. An electrochemical method for successively assessing the ability of each of
a plurality of candidate ligands to bind to a ligand-binding partner, comprising:
30 (a) providing a flow cell comprised of a detection chamber adapted to contain
in the range of about 0.5 jil to about 100 pi of liquid, an inlet for directing a stream of
liquid into the detection chamber, and an outlet for directing liquid out of the detection
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chamber, the detection chamber comprising a working electrode and a reference electrode,
and, disposed on the working electrode, a ligand-binding partner having an initial ligand
bound thereto and, attached to the initial ligand, a redox enzyme that catalyzes an
electrochemical reaction of a substrate, wherein the ability of a candidate ligand to •
5 displace the initial ligand from the ligand-binding partner corresponds to a decrease in the
rate of the rate of the electrochemical reaction;
(b) introducing about 0.5 \xl to about 100 jil of a sample through the inlet into
the detection chamber, wherein the sample is comprised of a liquid medium containing
about 0.1 pg to about 1 ng of a candidate ligand;
10 (c) measuring the rate of the electrochemical reaction;
(d) introducing a carrier liquid through the inlet into the detection chamber so
as to force the sample-containing liquid medium out of the detection chamber through the
outlet, and allowing flow of the carrier liquid through the detection chamber for a time at
least sufficient to ensure that the chamber is substantially free of the candidate ligand; and
15 (e) repeating steps (b), (c) and (d) with each of a plurality of candidate ligands
in succession, wherein each cycle comprised of steps (b), (c) and (d) has a cycle time not
exceeding about 30 seconds; and
(f) determining the ability of each candidate ligand to bind to the ligand- ■
binding partner from the measurements made in (c).
20
3. An electrochemical method for successively assessing the ability of each of
a plurality of candidate ligands to bind to a ligand-binding partner, comprising:
(a) providing a flow cell comprised of a detection chamber adapted to contain
in the range of about 0.5 jxl to about 100 jil of liquid, an inlet for directing a stream of
25 liquid into the detection chamber, and an outlet for directing liquid out of the detection
chamber, the detection chamber comprising a reference electrode and a working electrode
with the ligand-binding partner disposed thereon;
(b) introducing a sample through the inlet into the detection chamber, wherein
the sample is comprised of about 0.5 pi to about 100 pi of a liquid medium containing
30 about 0.1 pg to about 1 ng of a candidate ligand, the candidate ligand bound to a redox
enzyme that catalyzes an electrochemical reaction of a substrate of the redox enzyme;
(c) determining whether or not the electrochemical reaction is taking place of
at the working electrode;
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(d) introducing a carrier liquid into through the inlet and into the detection
chamber so as to force the sample-containing liquid medium out of the detection chamber
through the outlet, and allowing flow of the carrier liquid through the detection chamber
for a time at least sufficient to ensure that the chamber is substantially free of the
candidate ligand;
(e) repeating steps (b), (c) and (d) with each of a plurality of candidate ligands
in succession, wherein each cycle comprised of steps (b), (c) and (d) has a cycle time not
exceeding about 30 seconds; and
(f) determining the ability of each candidate ligand to bind to a ligand-binding
partner from the measurements made in (c).
4. The method of any one of claims 1 to 3, wherein the flow cell further
includes a redox mediator in the detection chamber.
5. The method of claim 4, wherein the redox mediator is disposed on the .
working electrode.
6. The method of claim 5, wherein the redox mediator is nonleachable.
7. The method of any of claims 1 to 3, wherein the volume of the sample is in
the range of about 0.5 to about 10 pi
8. The method of claim 7, wherein the volume of the sample is in the range of
about 0.5 to about 1 pi.
9. The method of any one of claims 1 to 3, wherein the quantity of candidate
compound or ligand in the sample is in the range of about 0. 1 pg to about 1 00 pg.
1 0. The method of claim 9, wherein the quantity of candidate compound or
ligand in the sample is in the range of about is in the range of about 0.1 pg to about 10 pg.
11. The method of claim 10, wherein the quantity of candidate compound or
ligand in the sample is in the range of about is in the range of about 0.1 pg to about 1 pg.
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12. The method of any one of claims 1 to 3, wherein the quantity of candidate
compound or ligand in the sample is at most about 10 pmoles.
5 13. The method of claim 1 2, wherein the quantity of candidate compound or
ligand in the sample is at most about 1 pmole.
14. The method of claim 13, wherein the quantity of candidate compound or
ligand in the sample is at most about 0.1 pmole.
10
1 5. The method of claim 1 , wherein each candidate compound is a
pharmacologically active agent
1 6. The method of either claim 2 or claim 3, wherein the sample further
15 comprises a substrate of the redox enzyme.
17. The method of any of claims 1 to 3, wherein step (c) comprises measuring
a current generated at the working electrode.
20 1 8. The method of claim 4, wherein the redox mediator provides electrical
contact between the target molecule and the working electrode.
19. The method of claim 4, wherein the redox mediator comprises a polymer
and a redox species ionically, covalently or coordinatively bound to the polymer.
25
20. The method of any one of claims 1 to 3, wherein the electrochemical ■
reaction takes place upon application of a potential between the working electrode and the
reference electrode.
30
21. The method of any one of claims 1 to 3, wherein the flow cell further
includes a third electrode.
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22. The method of any one of claims 1 to 3, wherein after step (b), flow
through the detection chamber is stopped, and not resumed until after step (c).
23. The method of any one of claims 1 to 3, wherein the working electrode
and the reference electrode are a facing electrode pair electrically insulated from each
other and separated by a predetermined distance.
24. The method of any one of claims 1 to 3, wherein the reference electrode is
positioned adjacent to the outlet, such that the reference electrode is in a downstream
position relative to the flow of sample through the detection chamber.
25. The method of any of one claims 1 to 3, wherein the exposed surface area
of the working electrode is less than about 10' 3 cm 2 .
26. The method of claim 25, wherein the exposed surface area of the working
electrode is less than about 10" 4 cm 2 .
27. The method of claim 26, wherein the exposed surface area of the working
electrode is less than about 10" 5 cm 2 .
28. The method of any one of claims 1 to 3, wherein the detection chamber is
adapted to contain in the range of about 0.5 to about 10 \d of liquid.
29. The method of claim 28, wherein the detection chamber is adapted to
contain in the range of about 0.5 to about 1 |xl of liquid.
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